X=ray diffraction and computer simulation techniques will be used to determine the three-dimensional structures and substrate recognition mechanisms of E. coli thioredoxin reductase and C. fasciculata trypanothione reductase, and to design improved inhibitors of trypanothione reductase. These dimeric enzymes are members of the widely distributed family of flavoprotein disulfide reductases that includes glutathione reductase, lipoamide dehydrogenase and mercuric reductase. Thioredoxin reductase differs from the others in having a protein substrate, thioredoxin, and is the one well characterized member of this family that has a sequence and active site that is fundamentally different from that of glutathione reductase, the model system for this enzyme family. Thioredoxin serves as a reductant of ribonucleotide reductase and plays various redox based regulatory roles in plant and animal cells. To understand the novel architecture of its reductase, and how it discriminates against small molecules in favor of thioredoxin, we have crystallized the enzyme and very recently determined its structure at 3 A resolution. This proposal aims at refining this preliminary structural model against available 2.7 A X-ray data, extending the resolution of the data (and the refinement) to 2 A or better, determining the structures of active site mutants, and of the enzyme co-crystallized with its substrates, thioredoxin and NADPH. The resulting structural detail will clarify the mechanism of the enzyme and nicely complement the knowledge of flavoprotein disulfide reductases that has been provided by glutathione reductase. Trypanothione reductase represents an enzyme target for drug intervention in African trypanosomiasis, Chagas' disease and leishmaniasis. The protozoan parasites do not possess glutathione reductase, and use a glutathione-based peptide, trypanothione, for the essential reduction of glutathione. Trypanothione reductase is highly homologous to glutathione reductase, but the two enzymes are mutually exclusive for substrate. We are using molecular dynamics simulations to predict the structure of trypanothione reductase from that of glutathione reductase. Our preliminary calculations help explain substrate specificity, but cannot proceed towards reliable evaluation of inhibitor binding without X-ray structures of the parasite enzyme. We have obtained crystals of the enzyme from an insect trypanosomatid that diffract to better than 3 A resolution, and we propose to solve the structure using molecular replacement. Cerami, Henderson and co-workers have developed inhibitors of the enzyme that when reduced by it undergo redox-cycling to produce toxic oxygen metabolites that kill the parasite. We propose to examine their binding modes by X-ray diffraction, and use the resulting structures as the basis for further molecular dynamics calculations aimed at improving these inhibitors.